RNA-seq of mutant embryos from the Mouse Genetics Project DMDD

Total RNA was extracted from embryonic lethal homozygous gene knockout and sibling embryos from the Mouse Genetics Project (http://www.dmdd.org.uk/). The 3 end of fragmented RNA was pulled down using polyT oligos attached to magnetic beads, reverse transcribed, made into Illumina libraries and sequenced using Illumina HiSeq paired-end sequencing. Protocol: Total RNA was extracted from mouse embryos and DNase treated. Fragmented RNA was enriched for the 3 ends by pulled down using an anchored polyT oligo attached to magnetic beads. An RNA oligo comprising part of the Illumina adapter 2 was ligated to the 5 end of the captured RNA and the RNA was eluted from the beads. Reverse transcription was primed with an anchored polyT oligo with part of Illumina adapter 1 at the 5 end followed by 4 random bases, then an A, C or G base, then one of twelve 5 base indexing tags and 14 T bases. An Illumina library with full adapter sequence was produced by 15 cycles of PCR. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/本数据集的样本取自小鼠遗传学项目（Mouse Genetics Project，http://www.dmdd.org.uk/）中的胚胎致死纯合基因敲除胚胎及其同窝胚胎，研究人员从这些样本中提取了总RNA（Total RNA）。将片段化RNA的3'端通过与磁珠偶联的寡聚dT寡核苷酸（polyT oligos）进行亲和富集捕获，随后进行反转录、构建Illumina测序文库（Illumina library），并采用Illumina HiSeq平台完成双端测序。 实验流程：从小鼠胚胎中提取总RNA，并进行脱氧核糖核酸酶（DNase）处理。通过与磁珠偶联的锚定寡聚dT寡核苷酸（anchored polyT oligo）富集片段化RNA的3'端；将包含部分Illumina接头2（Illumina adapter 2）序列的RNA寡核苷酸连接至捕获得到的RNA的5'端，随后将RNA从磁珠上洗脱下来。以锚定寡聚dT寡核苷酸作为反转录引物，该引物的5'端带有部分Illumina接头1（Illumina adapter 1）序列，其后依次连接4个随机碱基、1个A/C/G碱基、12种5碱基索引标签（indexing tags）中的任意一种以及14个胸腺嘧啶（T）碱基。通过15轮聚合酶链式反应（PCR）扩增，得到带有完整接头序列的Illumina测序文库。 本数据集属于预发布版本。若需了解惠康信托桑格研究所（Wellcome Trust Sanger Institute）共享的预发布数据的规范使用要求（含各类出版延期条款的详细说明），请访问http://www.sanger.ac.uk/datasharing/